The ultimate goal of the project is the development of a basis for successful therapy of solid human tumors with antifolates and a microbial enzyme isolated from a Flavobacterium sp. The value of the enzyme, a carboxypeptidase that hydrolyzes folates and antifolates (methotrexate and aminopterin), will depend upon its ability to ameliorate toxicity accompanying chemotherapy with antifolates without depressing anti-tumor activity. Current objectives deal with in-depth evaluation of high-purity carboxypeptidase (greater than 1000-fold purified). Studies now in progress and those proposed include detailed characterization in vitro, analysis of reactivity with methotrexate and the biologically active form of 5-methyltetrahydrofolate, and investigation of the effects of a variety of biochemical, chemical and physiological compounds and factors that may influence its activity in vivo. Evaluation in vivo will be conducted with the rat-Walker carcinosarcoma 256 experimental system. The efficiency of the carboxypeptidase to decrease plasma methotrexate concentrations will be explored over a range of steady state concentrations of methotrexate obtained with continuous infusion of drug. The information derived from these studies should describe the specificity, efficiency and reactivity, and other properties of the enzyme that may contribute to an understanding of its potential clinical value.